The intervertebral discs (IVDs) link adjacent vertebrae within the spine. They are composed of the peripheral annulus fibrosus (AF) and the central nucleus pulposus (NP). The AF is a fibrosus tissue with concentric lamellae rich in collagen fibrils (1). The NP has a more amorphous consistency, with collagen fibrils that have a random orientation and a high content of aggrecan that give it a gelatinous appearance and provides for the ability to resist compressive loads. Aggrecan is a large proteoglycan with numerous glycosaminoglycan (GAG) chains attached to its core protein, which in the NP provides the osmotic properties needed to counter the effects of compression.
Mechanisms that contribute to degenerative changes in the disc lead to biochemical alterations in the composition and structure of extracellular matrix due to both depleted synthesis and increased degradation, with aggrecan being particularly susceptible to proteolytic damage and loss. Aging, poor nutrition, biomechanical (2-5), biochemical (6-10) and genetic influences (11-14) are associated with increased IVD degeneration. During degeneration, loss of GAG content in the NP occurs, changing it from a gelatinous structure to a fibrotic texture as it becomes more collagenous, and fissures appear in both the NP and AF (15,16). This is commonly associated with low back pain, possibly due to the nerve ingrowth and loss of disc height, which are facilitated by proteoglycan depletion (17). Currently, there is no medical treatment for IVD degeneration, ultimately leaving surgical excision of the damaged tissue, insertion of a cage or prosthesis to restore the IVD space, and vertebral bone fusion as the only offered option. While this may provide relatively good clinical short-term results (18) in pain relief, in many instances it also alters spine biomechanics leading to subsequent adjacent-level disc degeneration.
Biological repair of the degenerating IVD would be preferable to surgical excision.
Disc degeneration starts early in life and progresses with increasing age (48, 49). This process is characterised by a phenotypic change of the resident cells and results in increased production of inflammatory cytokines (50, 51). A number of cytokines have been linked to disc degeneration; IL-1β and TNF-α were the first to be described, but additional candidates such as IL-6 and IL-8 have more recently been described especially in animal models (17). Studies of human discs from degenerate/herniated specimens showed, in addition to IL-13 and TNF-α, increased levels of IL-2, IL-4, IL-10, IL-12 and IL-17 when compared to healthy control (52). The exact mechanism leading to increased cytokine production is unclear. Multiple internal and external cues could influence cytokine production, such as heredity, mechanical loading, oxygenation, or the presence of inflammatory cells (17). In addition, accumulation of specific matrix fragmentation products may activate Toll-like receptors and thereby induce cytokine production.
Inflammatory cytokines are known to induce protease production, which subsequently leads to structural failure and loss of IVD height due to degradation of the extracellular matrix (ECM), including aggrecan and collagen (53). Although proteases are responsible for fragmentation and breakdown of important components of the ECM, they also have significant roles in normal remodeling of the disc. Cathepsin K activity, along with matrix metalloproteinase (MMP) proteolysis of aggrecan, has been suggested to be mainly a process of normal tissue remodeling in the disc (54, 55). However, matrix metalloproteinases (MMP1, 2, 3, 7, 9, 13), aggrecanases (ADAMTS4, 5), and cathepsins (cathepsins D and L) are all elevated during disc degeneration (56, 9). In addition, the serine protease HTRA1, is thought to play a central role in disc degeneration as elevated levels of HTRA1 and its degradation of CHAD correlated to the degree of disc degeneration (10, 57).
Degradation of the protein and proteoglycan content of the nucleus pulposus (NP) can result in loss of disc height and the weight bearing capacity of the disc. In the final stages of disc degradation fissures in the annular ring occur, leading to extrusion of NP material and pain due to compression of nerves. A repair strategy of the painful degenerate disc requires production of ECM components and down regulation of proteinase activity in the inflammatory milieu. These properties are associated with several growth factors such as TGF-β and BMP 7 (58-61). However, the use of growth factors in clinical practice is limited by their high cost and potential side effects.
Osteoarthritis (OA) is a chronic degenerative joint disorder that affects millions of people. It is characterized by the destruction of articular cartilage due to an imbalance in the anabolic and catabolic activities of chondrocytes. Articular cartilage is an avascular connective tissue, covering the bony parts of diarthrodial joints allowing the frictionless motion of the joint, by absorbing and dissipating load. These properties are related to the composition and structure of its extracellular matrix (ECM). It is composed of collagen fibrils, proteoglycans (predominantly aggrecan), noncollagenous proteins and a high content of water. The only cell type in articular cartilage is the chondrocyte, and is responsible for the synthesis and maintenance of the extracellular matrix.
During osteoarthritis (OA), characterized by degradation of articular cartilage and inflammation of the synovial membrane, this equilibrium is disrupted due to increased degradation of collagens and proteoglycans from the matrix and depleted synthesis of molecules. Cartilage responds to a complex multitude of autocrine and paracrine (anabolic and catabolic) factors that regulate gene expression and protein synthesis in chondrocytes.
Matrix degradation is mediated by matrix metalloproteinases (MMPs) and ADAMTS-4 and -5, induced by Interleukin-1beta (IL-1β) the major cytokine implicated in OA. Other cytokines that have been implicated in OA pathogenesis include tumor necrosis factor-alpha (TNF-α), IL-6, other common c-chain cytokines such as IL-2, IL-7, IL-15, and IL-21, and chemokines. These factors produced by synovial cells and chondrocytes results in the upregulation of members of the matrix metalloproteinase (MMP) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) families of enzymes. MMPs are involved in ECM turnover and cartilage degeneration. Aging, obesity, and joint injuries are associated with increased OA. It is characterized by progressive cellular and molecular changes in all joint tissues, including articular cartilage, subchondral bone, synovium, ligaments, and peri-articular muscles. There are currently no therapies that reverse or repair cartilage degradation in OA patients.
There is general agreement that since inflammatory processes play a fundamental role in the pathogenesis of various rheumatic diseases, such as, OA and rheumatoid arthritis (RA) selective inhibition of inflammatory activities is vital for therapy and that the family of NF-κB transcription factors play a prominent role in this process. Thus several studies have been directed towards the pharmacologic modulation of the NF-κB pathways using non-steroidal anti-inflammatory drugs, corticosteroids, nutraceuticals, antisense DNA therapy, RNA interference and anti-rheumatic drugs.
Link N is a 16 amino acid sequence that has been shown to increase proteoglycan synthesis and production of other matrix components by IVD cells (29, 34). It has also been shown to increase disc height in a rabbit disc puncture degeneration model, thereby demonstrating a regenerative potential also in vivo (31). This naturally occurring peptide represents the N-terminal region of the link protein that stabilizes proteoglycan aggregates in both disc and cartilage, and is generated by MMPs during tissue turnover in vivo. Link N interacts with the Bone Morphogenetic Protein (BMP) Type 11 Receptor and activates Smad1/5 signaling in cultured rabbit IVD cells (33).
Fragments of Link N have been tested. Wang et al. reported that the stimulatory effect of Link N was lost when they evaluated a number of shorter Link N-derived peptides (33) including a peptide spanning amino acid residues 1-12.